QSTORM-AO July 2019 Phone Meeting

July 16, 2019

by Carol Lynn Alpert

Participating:  Jessica, Peter, Abhijit, Abhilasha, Faiz, Carol Lynn, Kil Ho

Updates:  Kil Ho delivered a successful thesis defense, and he will be finishing up at OSU by the end of August.  Then he heads to Dow Chemical in Midland, Michigan, as Senior R&D Engineer in Process Development.  Congratulations Kil Ho!   

To Do’s:

Optics Team – Georgia

Abhijit has not posted an update since May 15, but promises to do so after this call. He and Peter have completed the first draft of their manuscript on imaging 100 nm polystyrene beads with holography.  Abhijit has set up the two-color imaging tools, and shown that they work optically.  However, the team is still encountering high background fluorescence limiting their ability to achieve high-resolution imaging of neuromuscular junctions in fruitflies with biologist Daichi Kamiyama. The growth cone filopedia on the neuro side of the junction is coming out sharp, but the filopedia on the muscle side are being drowned out by high background fluorescence, since the targeted molecule is not unique to the growth cone structure.  One technique is proving somewhat helpful: using a laser to bleach out everything surrounding the growth cone.  Next they will apply the two-color capacity using a splitter.  

The long-anticipated light sheet technique should also solve a number of problems, including stray light, and Abhijit is close to finishing a design for it on a CAD file simulator, which he will then probe for possible weaknesses.

QDot Team - Ohio

Faiz submitted a nicely written blog post on his and Abby's work. They are still trying to perfect the reversible quenching mechanism in DNA-Au-QDs.  (They've been able to quench, but not unquench.) They have ordered a new set of single strand DNA, designed to hit the desired melting point: below body temperature (37 C) on one side of the cis-trans conformation, but above it on the other.  Their new single strand design has azobenzene molecules on both of the complementary strands; 3 on one, 4 on the other, along with 10 nucleotides per strand.  The goal is to stabilize the DNA better in the trans form and destabilize it more in the cis form, enhancing the reversible switching mechanism. 

Meanwhile, Faiz conducted a neat experiment reproducing earlier results confirming the successful conjugation of QD-DNA-AuNP composites, with a corresponding 90% decrease in QD fluorescence.  Curiously, solutions with non-complementary strands quenched QDs even more than the complementary strands; this result could be due to random proximity of AuNPs to QDs in the solution, or to an “inner filter effect” in which the particles randomly absorb or scatter light meant for the QDs, or both.  In any case, it is a finding that will only appear in this kind of bulk testing scenario, not in the actual application, when excess Au will be washed from the samples.

Strawberries contain significant amounts of the aromatic organic dye Coumarin
Strawberries contain significant amounts
of the aromatic organic dye Coumarin 

Jessica is still waiting to hear about funding from NIH for continued work on the cage method of QDot quenching, though scores are high.  While she is developing the technique primarily for diagnostic use, she suspects it will also be useful for imaging.  Peter is going to select a cell membrane antigen target to image with green Coumarin 6 dye (excites at 420 or 450, emits at 500) for preliminary testing, and Jessica is going to work with Elizabeth to confirm high specific binding ratios before sending to Georgia.  Soon after, we hope to do a test using QDs in the cages for imaging.

 

Communications Team – Boston

Carol Lynn urged Jessica and Peter to move as quickly as possible on the imaging testing with the molecular cages, because it could become a centerpiece of the QSTORM film that has to be shot in January in order to complete post-production by April.   She would also like to have a conversation with Georgia bio collaborator Daichi Kamiyama and research assistant Melissa Inal to discuss their STORM imaging collaboration targeting neuromuscular junctions, and Peter said he would check with them on that.

Our next meeting is set for Aug 14 at 10 am.